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Chemokine-directed leukocyte migration is crucial for effective immune and inflammatory responses. Conventional chemokine receptors (cCKRs) directly control cell movement; atypical chemokine receptors (ACKRs) regulate coexpressed cCKRs; and both cCKRs and ACKRs internalize chemokines to limit their abundance in vivo, a process referred to as scavenging. A leukocyte's migratory and chemokine-scavenging potential is determined by which cCKRs and ACKRs it expresses, and by the ligand specificity, signaling properties, and chemokine internalization capacity of these receptors. Most chemokines can bind at least one cCKR and one ACKR. CCL2 can bind to CCR2 (a cCKR) and two ACKRs (ACKR1 and ACKR2). In this study, by using fluorescent CCL2 uptake to label cells bearing functional CCL2 receptors, we have defined the expression profile, scavenging activity, and ligand specificity of CCL2 receptors on mouse leukocytes. We show that qualitative and quantitative differences in the expression of CCR2 and ACKR2 endow individual leukocyte subsets with distinctive CCL2 receptor profiles and CCL2-scavenging capacities. We reveal that some cells, including plasmacytoid dendritic cells, can express both CCR2 and ACKR2; that Ly6Chigh monocytes have particularly strong CCL2-scavenging potential in vitro and in vivo; and that CCR2 is a much more effective CCL2 scavenger than ACKR2. We confirm the unique, overlapping, ligand specificities of CCR2 and ACKR2 and, unexpectedly, find that cell context influences the interaction of CCL7 and CCL12 with CCR2. Fluorescent chemokine uptake assays were instrumental in providing these novel insights into CCL2 receptor biology, and the sensitivity, specificity, and versatility of these assays are discussed. [ABSTRACT FROM AUTHOR]

Fluorescent activated cell sorting (FACS) is a powerful technique that allows rapid quantitative cell-by-cell analysis of the expression of multiple distinct proteins and the isolation of specific cell types from complex cellular mixtures. FACS-based detection of chemokine receptors is typically achieved using chemokine receptor-specific antibodies. However, this approach has some limitations, and, in our experience, many anti-chemokine receptor antibodies have poor signal-to-noise ratios and thus low sensitivity and reliability in FACS. Moreover, antibodies against some chemokine receptors are not commercially available, particularly in nonhuman species, and generating good antibodies against chemokine receptors is challenging and expensive. Here, we describe a simple alternative method of detecting chemokine receptors by FACS that relies on cellular internalization of fluorescently labelled chemokines. This approach detects chemokine receptors with high sensitivity, specificity, and reliability, and consistently outperforms antibody-based detection methods when applied to the analysis of mouse cells in our experience. It can also be used to study receptor specificity on primary cells and, since chemokines from one species often bind to receptors from other species, a single fluorescently labelled chemokine should allow reliable chemokine receptor detection and analysis across a range of species.